Human bispecific EGFRvIII antibody and CD3 engaging molecules

ABSTRACT

We have constructed bispecific antibody engaging molecules which have one arm that specifically engages a tumor cell which expresses the human EGFRvIII mutant protein on its surface, and a second arm that specifically engages T cell activation ligand CD3. The engaging molecules are highly cytotoxic and antigen-specific. These may be used as therapeutic agents.

This invention was made using funds from the U.S. government. The U.S.government retains certain rights in the invention according to theterms of NIH/NCI grant no. CA11898.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of cancer therapy. In particular,it relates to treating cancers that express EGFRvIII.

BACKGROUND OF THE INVENTION

The most common primary malignant brain tumor, glioblastoma multiforme(GBM), remains uniformly fatal despite surgical resection, radiationtherapy, and chemotherapy1. Immunotherapy promises to induce robust,tumor-specific immune responses that eliminate neoplastic cells withunparalleled specificity without adding additional toxicity tomultimodality therapy. Substantial evidence supports the role of T-cellsin the eradication of cancer. Recently, the concept of using specificantibodies to re-direct T-cells has been optimized in the form ofrecombinant bispecific T-cell engaging molecules, or bispecific T-cellengaging molecules, that consist of a tumor-targeting single-chainantibody connected to a single-chain antibody directed against a Tcellactivation ligand such as CD3. These bispecific T cell engagingmolecules can tether T-cells to tumor cells, which results in a highlylocalized and specific activation of T-cells with concomitant tumor celllysis. Recently, human trials using a CD19xCD3 bispecific T cellengaging molecule confirmed the potency of these constructs by tumorregression observed in 7/7 patients with non-Hodgkin's lymphoma at adose of only 0.06 mg/m2 with clearance of tumor from the blood, bonemarrow, and liver4. The most significant limitation of these promisingconstructs, however, is the lack of tumor-specific targets that arefrequently and homogeneously expressed.

Tumor-specific antigens derived from mutations in somatic genes are lesslikely to be associated with autoimmunity, but often arise randomly as aresult of the genetic instability of tumors and, as such, tend to bepatient-specific and incidental to the oncogenic process. EGFRvIII,however, is a frequent and consistent tumor specific mutation, centralto the neoplastic process, which consists of an in-frame deletion of 801base pairs from the extracellular domain (ECD) of the EGFR that splits acodon and produces a novel glycine at the fusion junction. This mutationencodes a constitutively active tyrosine kinase that enhances neoplasticcell growth and migration and confers radiation and chemotherapeuticresistance to tumor cells. The EGFRvIII mutation is most frequently seenin patients with GBM, but has been found in a broad array of othercommon cancers. The new glycine inserted at the fusion junction ofnormally distant parts of the ECD results in a tumor-specific epitope(FIG. 1) that is not found in any normal tissues.

There is a continuing need in the art to find better and more successfultreatments of cancers such as brain cancers.

SUMMARY OF THE INVENTION

One aspect of the invention is a bispecific polypeptide. The bispecificpolypeptide comprises a first human single chain variable region whichbinds to EGFRvIII. The first single chain variable region is in serieswith a second human single chain variable region. The second singlechain variable region binds to T cell activation ligand CD3. The firstsingle chain variable region comprises segments encoded by SEQ ID NO: 2and 3. The second single chain variable region comprises segmentsencoded by SEQ ID NO: 5 and 6.

Another aspect of the invention is a polynucleotide encoding thebispecific polypeptide. The bispecific polypeptide comprises a firsthuman single chain variable region which binds to EGFRvIII. The firstsingle chain variable region is in series with a second human singlechain variable region. The second single chain variable region binds toT cell activation ligand CD3. The first single chain variable regioncomprises segments encoded by SEQ ID NO: 2 and 3. The second singlechain variable region comprises segments encoded by SEQ ID NO: 5 and 6.

Another aspect of the invention is a method of treating anEGFRvIII-expressing tumor in a patient. The bispecific polypeptide isadministered to the patient, whereby a cytolytic T cell response to thetumor is induced. The bispecific polypeptide comprises a first humansingle chain variable region which binds to EGFRvIII. The first singlechain variable region is in series with a second human single chainvariable region. The second single chain variable region binds to T cellactivation ligand CD3. The first single chain variable region comprisessegments encoded by SEQ ID NO: 2 and 3. The second single chain variableregion comprises segments encoded by SEQ ID NO: 5 and 6.

Still another aspect of the invention is a method of making thebispecific polypeptide. A cell is cultured in a culture medium. The cellcomprises a polynucleotide encoding the bispecific polypeptide. Thebispecific polypeptide comprises a first human single chain variableregion which binds to EGFRvIII. The first single chain variable regionis in series with a second human single chain variable region. Thesecond single chain variable region binds to T cell activation ligandCD3. The first single chain variable region comprises segments encodedby SEQ ID NO: 2 and 3. The second single chain variable region comprisessegments encoded by SEQ ID NO: 5 and 6. After culturing, the bispecificpolypeptide is harvested from the cells or from the culture medium.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the wild-type EGFR and mutant EGFRvIII.PEPvIII: LEEKKGNYVVTDH (SEQ ID NO: 1)

FIG. 2 shows a model of an EGFR specific Fv-PEPvIII (SEQ ID NO: 1)complex.

FIG. 3A-3B shows antibody production from GBM patients receivingEGFRvIII-specific vaccination. FIG. 3A: Serum titers of GBM patientswere tested by ELISA against EGFRvIII-specific PEPvIII after eachvaccination. FIG. 3B: Binding of a patient serum sample to EGFRvIII ECDassayed by BIAcore analysis.

FIG. 4A-4C shows binding of EGFRvIII-specific antibodies fromimmortalized human B-cells derived from patients with GBM immunized witha peptide containing an EGFRvIII-specific epitope (PEPvIII). FIG. 4A(Left): Fluorescent cytometry analysis of supernatant derived from humanB-cells of a vaccinated GBM patient (ACT II-18) that were stimulatedwith PEPvIII on EGFRvIII-transfected 3T3 fibroblasts (NR6M) andwild-type EGFR transfectants (NR6W). FIG. 4B (Middle): Two weeks afterstimulation, supernatants are analyzed for specificity using NR6M cellsor NR6W cells. Supernatant from transformed human B-cells can produceantibodies recognizing NR6M cells. FIG. 4C (Right): E4, G5 and F12 weresingle-well picked from a 96-well plate of the patient sample Act II-18after B cells were enriched with a human B cell enrichment kit (StemCell #19054). Patient peripheral blood mononuclear cells are transformedwith 20% EBV virus (95.8 cell supernatant) in the presence of 10 mg/mLof cyclosporine. To improve the transformation efficiency, CpG 2006 wasadded at 2.5 ng/mL.

FIG. 5 is a flow diagram showing the generation of recombinant proteinMR1-1 scFv-antiCD3scFv-His6 (MR1-1 bispecific T cell engaging molecule).G4S=Gly4Ser; MR1-1xCD3 Bite show in SDS-PAGE.

FIG. 6A-6B shows the binding of MR1-1xCD3 bispecific T cell engagingmolecule. FIG. 6A (Left): Binding to NR6M and NR6W cells. As expected,MR1-1 shows some binding to wild-type EGFR at higher concentrations.FIG. 6B (Right): Binding of MR1-1-bispecific T cell engaging molecule tohuman GBM cell lines. U87MG.EGFR (wild type) (top) and U87MG.ΔEGFRvIII(bottom) stained with MR1-1xCD3 bispecific T cell engaging molecule.

FIG. 7 shows the binding of MR1-1xCD3 bispecific T cell engagingmolecule to CD3-expressing human PBMC by flow cytometry. In the toppanels, MR1-1 bispecific T cell engaging molecule was labeled with FITCand stained the PBMC cells directly together with anti-CD4 oranti-CD8-PE. In the bottom panels, binding of MR1-1 bispecific T cellengaging molecule to cells was detected by anti-His Alex 488.

FIG. 8A-8B shows MR1-1xCD3 mediates dose-dependent specific lysis invitro. Redirected lysis of glioma cell line U87MG (897) andU87MG-EGFRvIII (898) was tested with human PBL in the presence ofincreasing bispecific T cell engaging molecule concentrations for an18-h assay period. Effector and target cells were mixed at an E:T ratioof 20:1. Error bars indicate SD of triplicate measurements. Specificlysis was assessed via standard chromium release assay.

FIG. 9 shows dose-dependent inhibition of subcutaneous U87MG-EGFRvIIItumor growth in NSG mice by MR1-1xCD3. 3×10⁵ U87MG-EGFRvIII cells weremixed with 3×10⁵ human PBLs (E:T of 1:1) and subcutaneously injectedinto the right flanks of 8 male NSG mice per group. Treatment by tailvein injections with 1 μg, 10 μg, 100 μg, and vehicle control wasstarted 1 h after implantation of tumor cells. Treatment was repeatedfor four consecutive days. Tumors were measured three times a week withcalipers.

FIG. 10 is a flow diagram detailing the cloning of a fully humananti-EGFRvIII antibody from GBM patients.

FIG. 11 shows cDNA sequence and design for fully-human EGFRvIII-specificBiTE based on mAb 139 and 28F11. (139 VH; SEQ ID NO: 2); (139 VL; SEQ IDNO: 3); (Gly4Ser; SEQ ID NO: 4); (28F11 VH; SEQ ID NO: 5); (28F11 VL;SEQ ID NO: 6); (Hexahistidine; SEQ ID NO: 7)

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed human bispecific T cell engaging moleculeswhich target both the EGFRvIII and T cell activation ligand CD3. Theyhave been found to recruit cytotoxic T cells to a cancer cell expressingEGFRvIII and activate cytotoxic T cells, thereby killing the cancer cellexpressing the EGFRvIII molecule. The bispecific T cell engagingmolecules are selectively reactive with EGFRvIII and T cell activationligand CD3 displayed on the surface of mammalian cells which areaccessible to the antibody from the extracellular milieu.

Many types of bispecific antibodies can be constructed and used. Theseinclude, without limitation, quadroma-derived F(ab′)2, heterodimericscFv, heterodimeric Fab, diabodies, tandem diabodies, and tandem scFvmolecules. Bispecific antibodies can also be made using trifunctionalantibodies, i.e., antibodies that have a third specificity as well asthe initial two for EGFRvIII and a T cell activation ligand. The manyforms are well known in the art.

Once bispecific T cell engaging molecules have been constructed, theycan be produced in recombinant cells. Any suitable cell type can beused. If the bispecific T cell engaging molecules are secreted, they canbe harvested from the culture medium. If they remain intracellular, thecells can be collected and broken under suitable conditions to harvestthe bispecific T cell engaging molecules from the appropriate cellfraction. Any convenient cell host can be used for producing thebispecific T cell engaging molecules, including bacteria, yeast, insectcells, plant cells, algal cells, mammalian cells. In one scenario, thebispecific T cell engaging molecules can be produced in stablytransfected CHO cells and the supernatant will contain the producedbispecific T cell engaging molecules.

Any tumor which expresses EGFRvIII can be targeted and treated with thebispecific T cell engaging molecules. Tumors which have been found toexpress the EGFRvIII antigen include brain tumors such as glioblastomamultiforme, breast tumors, and lung tumors. Any of these or other tumorscan be targeted if it expresses the mutant antigen.

DEFINITIONS

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; amino acid sequencesare written left to right in amino to carboxy orientation. The headingsprovided herein are not limitations of the various aspects orembodiments of the disclosure which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

“EGFRvIII” means a mutant form of the epidermal growth factor receptorrecognized by MR1 scFv and characterized by an 801 base pair in framedeletion of exons 2 to 7 near the amino terminal. This form of thereceptor is known in the art, as exemplified by the Wickstrand et al.,Moscatello et al., and Lorimer et al. references cited in theBackground. Due to a change in terminology, EGFRvIII was originallytermed a Type II mutation in some earlier work in the field, asexemplified by U.S. Pat. No. 5,212,290.

The term “CD3” refers to the protein complex associated with the T cellreceptor. Antibodies directed against CD3 are able to generate anactivation signal in T lymphocytes. Other T cell activation ligands canbe used as well, including without limitation CD28, CD134, CD137, andCD27.

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies), heteroconjugate antibodies (e.g.,bispecific antibodies) and recombinant single chain Fv fragments (scFv),disulfide stabilized (dsFv) Fv fragments (See, U.S. Ser. No. 08/077,252,incorporated herein by reference), or pFv fragments (See, U.S.Provisional Patent Applications 60/042,350 and 60/048,848, both of whichare incorporated herein by reference.). The term “antibody” alsoincludes antigen binding forms of antibodies (e.g., Fab′, F(ab′)2, Fab,Fv and rIgG (See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.).

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors (See, e.g., Huse, etal., Science 246:1275-1281 (1989); Ward, et al., Nature 341:544-546(1989); and Vaughan, et al., Nature Biotech. 14:309-314 (1996)).

Typically, an immunoglobulin has a heavy and light chain. Each heavy andlight chain contains a constant region and a variable region. Light andheavy chain variable regions contain a “framework” region interrupted bythree hypervariable regions, also called complementarity-determiningregions or CDRs. The extent of the framework region and CDRs have beendefined (see. SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST. Kabat,E., et al., U.S. Department of Health and Human Services, (1987); whichis incorporated herein by reference). The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDRs in three dimensional space. TheCDRs are primarily responsible for binding to an epitope of an antigen.The CDRs are typically referred to as CDR1, CDR2, and CDR3, numberedsequentially starting from the N-terminus.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe heavy chain and the light chain of a traditional two chain antibodyhave been joined to form one chain. Typically, a linker peptide isinserted between the two chains to allow for proper folding and creationof an active binding site.

The term “linker peptide” includes reference to a peptide within anantibody binding fragment (e.g., Fv fragment) which serves to indirectlybond the variable heavy chain to the variable light chain. The linkermay be a series of a single amino acid or an alternating pattern ofamino acids, for example.

The term “contacting” includes reference to placement in direct physicalassociation. With regards to this invention, the term refers toantibody-antigen binding.

As used herein, the term “bispecific T-cell engaging molecule” refers toa molecule designed to harness a subject's T cells to kill cancer cellsby targeting to the tumor cells expressing a desired molecule. Incertain embodiments, the desired molecule is human EGFRvIII. In otherembodiments, the bispecific T cell engaging molecules comprises two Fvdomains. In other embodiments, the bispecific T cell engaging moleculecomprises a first Fv domain directed to EGFRvIII and a second Fv domaindirected to CD3. The Fv domains may be scFv domains.

The term “selectively reactive” includes reference to the preferentialassociation of an antibody, in whole or part, with a cell or tissuebearing EGFRvIII or CD3 and not to cells or tissues lacking EGFRvIII orCD3. It is, of course, recognized that a certain degree of non-specificinteraction may occur between a molecule and a non-target cell ortissue. Nevertheless, selective reactivity, may be distinguished asmediated through specific recognition of EGFRvIII and CD3. Althoughselectively reactive antibodies bind antigen, they may do so with lowaffinity. On the other hand, specific binding results in a much strongerassociation between the antibody and cells bearing EGFRvIII or CD3 thanbetween the bound antibody and cells lacking EGFRvIII or CD3 or lowaffinity antibody-antigen binding. Specific binding typically results ingreater than 2-fold, preferably greater than 5-fold, more preferablygreater than 10-fold and most preferably greater than 100-fold increasein amount of bound antibody (per unit time) to a cell or tissue bearingEGFRvIII or CD3 as compared to a cell or tissue lacking EGFRvIII or CD3.Specific binding to a protein under such conditions requires an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats are appropriate for selecting antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow &Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications,New York (1988), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity. In someembodiments of the invention the antibody will bind to EGFRvIII betterthan to wild-type EGFR. In some instances an antibody will bind to both.The differential binding may be reflected in a stronger binding, or in afaster binding, or in more binding to a fixed amount of antigen with afixed amount of time. The better binding may be by a factor of at least2, 4, 6, 8, or 10. Under some disease conditions, it may be advantageousto have some degree of binding to both mutant and wild-type forms ofEGFR, for example where both forms are co-expressed on a tumor target.Under other disease situations, it may be desirable to have the maximumamount of specify available for the mutant form, for example, to reduceadverse side effects.

As used herein, “polypeptide,” “peptide,” and “protein” are usedinterchangeably and include reference to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers. The terms also apply to polymers containing conservativeamino acid substitutions such that the protein remains functional.

The term “residue” or “amino acid residue” or “amino acid” includesreference to an amino acid that is incorporated into a protein,polypeptide, or peptide (collectively “peptide”). The amino acid can bea naturally occurring amino acid and, unless otherwise limited, canencompass known analogs of natural amino acids that can function in asimilar manner as naturally occurring amino acids.

The phrase “disulfide bond” or “cysteine-cysteine disulfide bond” refersto a covalent interaction between two cysteines in which the sulfuratoms of the cysteines are oxidized to form a disulfide bond. Theaverage bond energy of a disulfide bond is about 60 kcal/mol compared to1-2 kcal/mol for a hydrogen bond. In the context of this invention, thecysteines which form the disulfide bond are within the framework regionsof the single chain antibody and serve to stabilize the conformation ofthe antibody. Cysteine residues can be introduced, e.g., by sitedirected mutagenesis, so that stabilizing disulfide bonds can be madewithin the molecule.

As used herein, “recombinant” includes reference to a protein producedusing cells that do not have, in their native state, an endogenous copyof the DNA able to express the protein. The cells produce therecombinant protein because they have been genetically altered by theintroduction of the appropriate isolated nucleic acid sequence. The termalso includes reference to a cell, or nucleic acid, or vector, that hasbeen modified by the introduction of a heterologous nucleic acid or thealteration of a native nucleic acid to a form not native to that cell,or that the cell is derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell, express mutants of genes that arefound within the native form, or express native genes that are otherwiseabnormally expressed, under expressed or not expressed at all.

As used herein, “nucleic acid” or “nucleic acid sequence” includesreference to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence includesthe complementary sequence thereof as well as conservative variants,i.e., nucleic acids present in wobble positions of codons and variantsthat, when translated into a protein, result in a conservativesubstitution of an amino acid.

As used herein, “encoding” with respect to a specified nucleic acid,includes reference to nucleic acids which comprise the information fortranslation into the specified protein. The information is specified bythe use of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolum (Proc. Nat'l Acad.Sci. USA 82:2306-2309 (1985), or the ciliate Macronucleus, may be usedwhen the nucleic acid is expressed in using the translational machineryof these organisms.

The phrase “fusing in frame” refers to joining two or more nucleic acidsequences which encode polypeptides so that the joined nucleic acidsequence translates into a single chain protein which comprises theoriginal polypeptide chains.

As used herein, “expressed” includes reference to translation of anucleic acid into a protein. Proteins may be expressed and remainintracellular, become a component of the cell surface membrane or besecreted into the extracellular matrix or medium.

By “host cell” is meant a cell which can support the replication orexpression of the expression vector. Host cells may be prokaryotic cellssuch as E. coli, or eukaryotic cells such as yeast, insect, amphibian,or mammalian cells.

The phrase “phage display library” refers to a population ofbacteriophage, each of which contains a foreign cDNA recombinantly fusedin frame to a surface protein. The phage displays the foreign proteinencoded by the cDNA on its surface. After replication in a bacterialhost, typically E. coli, the phage which contain the foreign cDNA ofinterest are selected by the expression of the foreign protein on thephage surface.

“Sequence identity” in the context of two nucleic acid or polypeptidesequences includes reference to the nucleotides (or residues) in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window. When percentage of sequence identityis used in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. Where sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17(1988), e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA). An indication that two peptide sequencesare substantially similar is that one peptide is immunologicallyreactive with antibodies raised against the second peptide. Thus, apeptide is substantially similar to a second peptide, for example, wherethe two peptides differ only by a conservative substitution.

A “comparison window”, as used herein, includes reference to a segmentof about 10-20 residues in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison may be conducted by the local homology algorithm of Smith& Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignmentalgorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970); by thesearch for similarity method of Pearson & Lipman, Proc. Nat'l Acad. Sci.USA 85:2444 (1988); by computerized implementations of these algorithms(including, but not limited to CLUSTAL in the PC/Gene program byIntelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST. FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), Madison, Wis., USA); the CLUSTAL program is well describedby Higgins & Sharp, Gene 73:237-244 (1988) and Higgins & Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucl. Acids Res. 16:10881-90 (1988);Huang, et al., Computer Applications in the Biosciences 8:155-65 (1992);and Pearson, et al., Meth. in Molec. Biol. 24:307-31 (1994).

The terms “effective amount” or “amount effective to” or“therapeutically effective amount” include reference to a dosage of atherapeutic agent sufficient to produce a desired result, such asinhibiting cell protein synthesis by at least 50%, or killing the cell.

The term “therapeutic agent” includes any number of compounds currentlyknown or later developed to act as anti-neoplastics,anti-inflammatories, cytokines, anti-infectives, enzyme activators orinhibitors, allosteric modifiers, antibiotics or other agentsadministered to induce a desired therapeutic effect in a patient.

The term “in vivo” includes reference to inside the body of the organismfrom which the cell was obtained. “Ex vivo” and “in vitro” means outsidethe body of the organism from which the cell was obtained.

Once the nucleic acids encoding a bispecific T cell engaging molecule ofthe present disclosure are isolated and cloned, one may express thedesired protein in a recombinantly engineered cell such as bacteria,plant, yeast, insect, and mammalian cells. It is expected that those ofskill in the art are knowledgeable in the numerous expression systemsavailable for expression of proteins including E. coli, other bacterialhosts, yeast, and various higher eucaryotic cells such as the COS, CHO,HeLa and myeloma cell lines. No attempt to describe in detail thevarious methods known for the expression of proteins in prokaryotes oreukaryotes will be made. In brief, the expression of natural orsynthetic nucleic acids encoding the isolated proteins of the inventionwill typically be achieved by operably linking the DNA or cDNA to apromoter (which is either constitutive or inducible), followed byincorporation into an expression cassette. The cassettes can be suitablefor replication and integration in either prokaryotes or eukaryotes.Typical expression cassettes contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the DNA encoding the protein. To obtain high levelexpression of a cloned gene, it is desirable to construct expressioncassettes which contain, at the minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation, anda transcription/translation terminator. For E. coli this includes apromoter such as the T7, trp, lac, or lambda promoters, a ribosomebinding site and preferably a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter andpreferably an enhancer derived from immunoglobulin genes, SV40,cytomegalovirus, and a polyadenylation sequence, and may include splicedonor and acceptor sequences. The cassettes of the invention can betransferred into the chosen host cell by well-known methods such ascalcium chloride transformation or electroporation for E. coli andcalcium phosphate treatment, electroporation or lipofection formammalian cells. Cells transformed by the cassettes can be selected byresistance to antibiotics conferred by genes contained in the cassettes,such as the amp, gpt, neo and hyg genes. It is also contemplated thatthe DNA can be delivered to a recipient patient, for example, onnanoparticles or other DNA delivery system, and that the patient mayproduce her own bispecific T-cell engaging molecules in vivo.

One of skill would recognize that modifications can be made to a nucleicacid encoding a polypeptide of the present disclosure (i.e.,anti-EGFRvIII or anti-CD3) without diminishing its biological activity.Some modifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

The phrase “malignant cell” or “malignancy” refers to tumors or tumorcells that are invasive and/or able to undergo metastasis, i.e., acancerous cell.

In addition to recombinant methods, the bispecific T cell engagingmolecules of the present disclosure can also be constructed in whole orin part using standard peptide synthesis. Solid phase synthesis of thepolypeptides of the present invention of less than about 50 amino acidsin length may be accomplished by attaching the C-terminal amino acid ofthe sequence to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, THE PEPTIDES: ANALYSIS,SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE SYNTHESIS. PARTA. pp. 3-284; Merrifield, et al. J. Am. Chem. Soc. 85:2149-2156 (1963),and Stewart, et al., SOLID PHASE PEPTIDE SYNTHESIS, 2ND ED., PierceChem. Co., Rockford, Ill. (1984). Proteins of greater length may besynthesized by condensation of the amino and carboxyl termini of shorterfragments. Methods of forming peptide bonds by activation of a carboxylterminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

In addition to redirecting T-cells to tumor-specific antigens, thebispecific T cell engaging molecules can also be used to carry otherdiagnostic or therapeutic compounds to cells expressing EGFRvIII ontheir surface. Thus, a bispecific T cell engaging molecule may beattached directly or indirectly, e.g., via a linker, to a drug so thatit will be delivered directly to cells bearing EGFRvIII. Therapeuticagents include such compounds as nucleic acids, proteins, peptides,amino acids or derivatives, glycoproteins, radioisotopes, lipids,carbohydrates, or recombinant viruses. Nucleic acid therapeutic anddiagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides.

Alternatively, the molecule linked to the bispecific T cell engagingmolecule may be an encapsulation system, such as a liposome or micellethat contains a therapeutic composition such as a drug, a nucleic acid(e.g. an antisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735;and Connor, et al., Pharm. Ther. 28:341-365 (1985).

The bispecific T cell engaging molecules of this disclosure areparticularly useful for parenteral administration, such as intravenousadministration or administration into a body cavity or lumen of anorgan. For example, ovarian malignancies may be treated by intravenousadministration or by localized delivery to the tissue surrounding thetumor. For treatment of tumors in the brain, the molecules may bedelivered directly to the brain, for example by injection or themolecules can be administered intravenously and then cross the bloodbrain barrier.

The compositions for administration will commonly comprise a solution ofthe bispecific T cell engaging molecules dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of fusion protein in these formulations can vary widely,and will be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs.

Thus, a typical pharmaceutical composition of the present invention forintravenous administration would be about 0.1 to 10 mg per patient perday. Dosages from 0.1 up to about 100 mg per patient per day may beused, particularly if the drug is administered to a secluded site andnot into the circulatory or lymph system, such as into a body cavity orinto a lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as REMINGTON'SPHARMACEUTICAL SCIENCE, 19TH ED., Mack Publishing Company, Easton, Pa.(1995).

The compositions of the present invention can be administered fortherapeutic treatments. In therapeutic applications, compositions areadministered to a patient suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. An effective amount of the compound is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the proteins of this invention to effectively treat thepatient. Preferably, the dosage is administered once but may be appliedperiodically until either a therapeutic result is achieved or until sideeffects warrant discontinuation of therapy. Generally, the dose issufficient to treat or ameliorate symptoms or signs of disease withoutproducing unacceptable toxicity to the patient.

Controlled release parenteral formulations of the pharmaceuticalcompositions of the present disclosure can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., THERAPEUTIC PEPTIDES AND PROTEINS:FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein as a central core. In microspheres the therapeuticis dispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 μm are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively. Capillarieshave a diameter of approximately 5 μm so that only nanoparticles areadministered intravenously. Microparticles are typically around 100 μmin diameter and are administered subcutaneously or intramuscularly. See,e.g., Kreuter. J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed.,Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice &Tabibi, TREATISE ON CONTROLLED DRUG DELIVERY, A. Kydonieus, ed., MarcelDekker, Inc. New York. N.Y., pp. 315-339 (1992) both of which areincorporated herein by reference.

Polymers can be used for ion-controlled release of pharmaceuticalcompositions of the present invention. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)).For example, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston, etal., Pharm. Res. 9:425-434 (1992); and Pec, et al., J. Parent. Sci.Tech. 44(2):58-65 (1990)). Alternatively, hydroxyapatite has been usedas a microcarrier for controlled release of proteins (Ijntema, et al.,Int. J. Pharm. 112:215-224 (1994)). In yet another aspect, liposomes areused for controlled release as well as drug targeting of thelipid-capsulated drug (Betageri, et at., LIPOSOME DRUG DELIVERY SYSTEMS,Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Numerousadditional systems for controlled delivery of therapeutic proteins areknown. See, e.g., U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871,4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,514,670;5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961;5,254,342 and 5,534,496, each of which is incorporated herein byreference.

Among various uses of the bispecific T cell engaging molecules of thepresent invention are included a variety of disease conditions caused byspecific human cells that may be eliminated by the toxic action of thefusion protein. One preferred application for the bispecific T cellengaging molecules of the invention is the treatment of malignant cellsexpressing EGFRvIII. Exemplary malignant cells include astrocytomas,glioblastomas, melanoma and the like.

As used herein, “mammalian cells” includes reference to cells derivedfrom mammals including humans, rats, mice, guinea pigs, chimpanzees, ormacaques. The cells may be cultured in vivo or in vitro.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLES Example 1

We constructed the molecule MR1-1XaCD3, which consists of MR1-1, themurine anti-human EGFRvIII single-chain Fv, and aCD3, the murineanti-human CD3 single-chain Fv. MR1-1XaCD3 was expressed in and purifiedfrom bacteria BL21 (DE3), and the activity of this double functionmolecule was confirmed by FACS showing its specific binding toEGFRvIII-expressing cell lines, as well as human T cells. Thecytotoxicity of MR1-1XaCD3 on EGFRvIII-expressing GBM D54MG.EGFRvIIIcell lines was measured in vitro by standard chromium release assay. Theefficacy of MR1-1XaCD3 was evaluated in NOD/SCID gamma mice where humanEGFRvIII-expressing cell lines were implanted. Our results showed thatthe MR1-1xaCD3 construct is highly cytotoxic and antigen-specific, withan 8-fold increase in specific lysis for D54MG.EGFRvIII over thewild-type control. In a subcutaneous model, tumor growth was inhibitedat a dose dependent manner. While total inhibition was achieved at the100 mcg/mouse/day, low dose might work effectively upon optimization. Insummary, our experiments showed that a human EGFRvIII-specific T-cellengaging molecules, MR1-1XaCD3, is effective on EGFRvIII-expressingtumor.

A murine MAb scFv MR1-1 (FIG. 2), specific for EGFRvIII, has beendemonstrated to be a suitable vehicle for GBM treatment in the format ofrecombinant immunotoxins, which is now in a clinical trial at ourinstitution (BB-IND-12,589). However, the innate properties ofmurine-derived antibodies might induce neutralizing antibodies thatlimit their wider application. Therefore, fully human MAbs are moredesirable for the construction of recombinant bispecific T cell engagingmolecules for clinical trials.

To obtain high-affinity human anti-EGFRvIII MAbs and scFvs we will (1)fuse EGFRvIII-specific B-cells to the HMMA2.5 non-secreting myelomapartner using an electrofusion technique or (2) clone the variable heavyand light chains from DNA libraries prepared from the antibody-secretingB-cell clones derived directly from GBM patients who have beenvaccinated with an EGFRvIII-specific epitope.

In the context of an ongoing clinical trial in these patients, we haveshown that vaccination with an EGFRvIII-specific peptide (PEPvIII:LEEKKGNYVVTDH; SEQ ID NO: 1) induced high-titer antibodies specific toEGFRvIII in 32 of 43 patients, with some patients developingtiters >1:2,000,000 (FIG. 3A, Left). Production of high-affinityEGFRvIII-specific antibodies with an average of 6 nM (KD) was confirmedby analyzing EGFRvIII ECD using BIAcore SPR analysis (FIG. 3B, Right).

It has often been challenging to clone MAbs directly from human B cellsin an efficient way. In our preliminary results, the supernatant derivedfrom human B-cells of a vaccinated GBM patient (ACT II-18 in FIG. 4)after stimulation with PEPvIII demonstrated positive reactivity onEGFRvIII transfected cells (NR6M) and wild-type EGFRwt transfectants(NR6W) at later days (FIG. 4A, Left). With samples from our patients, wewere able to transform B-cells with Epstein-Barr virus (EBV), and wedemonstrated that these cells maintain their ability to secretehigh-titer, EGFRvIII-specific antibodies after peptide stimulation forprolonged periods of at least 2 weeks (FIG. 4B, Middle). In our pilotstudies, three out of four patient samples have been successfullytransformed and demonstrated high-titer and high-affinity antibodyproduction (FIG. 4C. Right).

Example 2

To construct a recombinant bispecific T cell engaging molecule based onthese human EGFRvIII-specific scFvs, we will subclone the humananti-EGFRvIII scFvs into an existing cassette, which we previously usedto create an MR1-1xCD3 molecule (FIG. 5), by substituting the MR1-1scFvportion. Mouse anti-human CD3 scFv was cloned from a hybridoma line OKT3(ATCC, CRL 8001). MR1-1 bispecific T cell engaging protein afterpurification was shown in SDS-PAGE gel, with a molecular weight of 55kDa (FIG. 5; inset). The MR1-1 bispecific T cell engaging molecule bindsto EGFRvIII specifically and engages T-cells concomitantly throughbinding to CD3 (FIG. 6). EGFRvIII binding capacity and specificity wasconfirmed by using NR6M (EGFRvIII) and NR6W (EGFRwt) cell lines and aGBM cell line transfected with EGFRvIII (U87MG.ΔEGFR) (FIG. 6).Similarly, binding to CD3 was confirmed by staining human peripheralblood mononuclear cells (PBMCs) and Jurkat cells, showing the co-bindingof either anti-CD4 or anti-CD8 with bispecific T cell engaging moleculeson the same T-cell subpopulation from human PBMC or Jurkat cells (FIG.7). Although using human scFvs to generate bispecific T cell engagingmolecules may reduce the generation of neutralizing antibodies andpermit repeated administrations, the existing MR1-1 bispecific T cellengaging molecule may also be used.

Example 3

In order to minimize potential allogeneic responses against the tumorcells, we used our bank of existing matched human peripheral bloodlymphocytes (PBLs) and GBM cell lines in these assays. Cytotoxicity ofthe MR1-1 bispecific T cell engaging molecule was measured by a standardchromium-release assay using unstimulated PBLs as effector cells andhuman GBM cell line U87MG, which expresses wild-type EGFR, and thetransfected U87MG-EGFRvIII cell line as target cells. Results show thatthe MR1-1 construct is highly cytotoxic and antigen-specific, with anearly 25-fold increase in specific lysis (%) for U87MGEGFRvIII over thewild-type control, U87MG (FIG. 8A, left). These results echo, if notexceed, the findings that were initially reported in vitro forbscCD19xCD34, a bispecific construct that has since been tested in humantrials and found to induce potent tumor regression in patients withnon-Hodgkin's lymphoma. The specific lysis by MR1-1 bispecific T cellengaging molecule was at 30% of U87 MG-EGFRvIII compared to ˜1%-2% ofcontrol cells at 18 h (FIG. 8B, right), showing MR1-1 bispecific T cellengaging molecule mediated dose dependent specific lysis in vitro.

Example 4

The efficacy of MR1-1 bispecific T cell engaging molecule was evaluatedin NSG mice. We have determined the MR1-1 bispecific T cell engagingmolecule efficacy against U87MGEGFRvIII in NSG mice s.c. Briefly, U87MG-EGFRIII cells (70%-80% confluence) were harvested with 0.25%Trypsin-EDTA. Cells were washed 2× with sterile PBS. PBLs were harvestedas the non-adherent portion from healthy donor PBMC leukaphereses aftera 1 h incubation in AIM-V 2% HABS. Three ×105 U87MG-EGFRvIII cells weremixed with 3×105 human PBLs (E:T of 1:1) and injected s.c. into theright flanks of 8 male NSG mice per group. Treatment by tail veininjections with 1 μg, 10 μg, 100 μg, and vehicle control was started 1 hafter implantation of tumor cells. Treatment was repeated for fourconsecutive days. The results showed that the inhibition of subcutaneousU87MG-EGFRvIII tumor growth in NSG mice by MR1-1xCD3 was dose-dependentafter 28 days of observation (FIG. 9). Untreated tumor continued to growsteadily. There were two palpable tumors out of 8 in the 1-μg group.There was one palpable tumor in the 10-μg treated group. There were notumors in the 100-μg treated group. The efficacy of MR1-1 bispecific Tcell engaging molecule on the treatment of U87MG-EGFRvIII was verysignificant and encouraging in terms of the potency and dose dependency.

Example 5

EBV-transformed B cells provide only a transient reservoir ofmulti-clonal anti-EGFRvIII antibodies due to the high instability ofviral incorporation into the human genome. To make stable lines thatsecrete antibody and to clone EGFRvIII-specific scFvs from thesesamples, we will use human hybridoma technology and electrofusion aspreviously described13. Briefly. EBV-transformed PBMCs will be fusedwith hetermyeloma cell line HMMA2.5 by using a CytoPulse HybrimuneElectrofusion System (Cytopulse). Single cell clones will be screened byHAT (hypoxanthine, aminopterin, and thymidine) selection and confirmedby ELISA of supernatant against recombinant EGFRvIII ECD, or by flowcytometry against EGFRvIII expression cells lines and a cocktail ofB-cell markers. Alternatively, we will construct a phage scFv displaylibrary expressing human immunoglobulin genes that can be screened onEGFRvIII ECD, and affinity-maturation will be done when needed, aspreviously described. Another approach will be to bypass the screeningstep by using high-throughput DNA sequencing and bioinformatic analysisto mine antibody variable region (V)-gene repertoires from plasma cellsas described19. VH and VL CDRs of the selected EGFRvIII-specificMAb-expressing hybridomas will be amplified by using isotype-specificprimers, and scFv will be constructed in which the scFv protein has beentagged at the carboxy terminus with the hexahistidine sequences forpurification and detection. Expression, production, and characterizationof scFv will be carried out as described by using a metal affinitycolumn. Binding of scFv will be confirmed by flow cytometry on cellsexpressing EGFRvIII. At least one fully human anti-EGFRvIII MAb will beisolated and its scFv is expected to have an affinity higher than MR1-1(1.5 nM).

Example 6

To make the whole anti-EGFRvIII bispecific T cell engaging moleculehuman, we will generate a human scFv mimotope of murine MAb OKT3 thatreacts with CD3 antigen via screening from a human scFv phage displaylibrary. The human scFv phagemid library21, obtained from Los AlamosNational Laboratory, has very high size (7.1×1013 pfu/mL) and diversity(3×1011). During selection, the biotinylated target antigen, CD3 (SinoBiological Inc.), is incubated with the scFv library, and complexesformed are captured upon magnetic streptavidin-coated beads. Thebead+Ag/scFv complex is washed to remove nonspecific or low-affinitybinding phage. The bead+Ag/scFv complex is treated with acid (0.1M HCl)to recover all scFvs that bind to the target antigen. To recover mimicsof the mouse antibodies, the bead+Ag/scFv complex will be incubated withthe corresponding mouse OKT3 IgG for competition/elution of the bindingscFv antibodies. To generate those scFv antibodies with the highestaffinity, the selection pressure will be increased through each roundfor 3 rounds. Once scFvs have been recovered that bind CD3, competitionELISA will be carried out to determine whether they are true mimics ofthe mouse OKT3, and affinity-maturation will be done if needed. We willthen determine the T-cell activation function of 7 the human versionOKT3 of anti-CD3 scFv by carrying out (1) T-lymphocyte proliferationassays, (2) cytokine release assays, and (3) detection of the expressionof early T-cell activation marker by FACS as described22.

Example 7

We will construct a human anti-EGFRvIII scFv and a human anti-CD3 scFvby linking VH and VL fragments with a (Gly4Ser)3 peptide linker. Ahexahistidine tag was introduced at the C-terminus of human anti-CD3scFv to assist the detection and purification. Expression andpurification of the new fully human anti-EGFRvIII bispecific T cellengaging molecule will follow the same protocol as described above forMR1-1 bispecific T cell engaging molecule according to our previousprotocol.

Example 8

Building upon these promising preliminary data from our MR1-1 bispecificT cell engaging molecule studies, we will assess the cytotoxic activityof new fully human EGFRvIII-targeted bispecific T cell engagingmolecules by following the same protocol as described above. Negativecontrol experiments will be carried out with medium instead ofbispecific T cell engaging molecule or effector cells. Specific lysiswill then be calculated as [(cpm, experimental release)−(cpm,spontaneous release)]/[(cpm, maximal release)−(cpm, spontaneousrelease)].

Example 9

The efficacy of novel fully human EGFRvIII bispecific T cell engagingmolecules will be evaluated in NSG mice as described previously, as wellas in the preliminary studies. Our program has at its disposal a numberof EGFRvIII-expressing human GBM xenografts and cell lines with matchedautologous lymphocytes cryogenically preserved. Briefly, prior tobispecific T cell engaging molecule administration, tumor cells andlymphocytes will be mixed at a ratio of 1:1 and implanted in the caudatenucleus of the NSG mice using a Kopf stereotactic frame as we previouslydescribed24. The NSG mouse model will provide “proof-of-concept”efficacy studies against a GBM expressing EGFRvIII in the CNS. Prior tobeginning efficacy experiments, however, a maximum tolerated dose (MTD)of each candidate bispecific T cell engaging molecule will beestablished in these mice. Individual cohorts of 40 animals each will beadministered a candidate molecule, with doses between groups increasedby one half log 10 from 0.001 to 1 μg until an MTD is established. Onthe basis of prior work, mice will be treated intravenously (i.v.) withdaily bispecific T cell engaging molecule doses for 5 days. Therapy atthe MTD will start after approximately one-third of the median survivaltime has elapsed according to our prior experience with these tumors.Treatment will consist of the i.v. administration of the bispecific Tcell engaging molecule construct at predetermined doses.

Example 10

Fully Human BiTE Design—We have generated cDNA encoding a fully humanEGFRvIII-specific BiTE designated 139x28F11 (FIG. 11). The mAbs 139 (US2010/0111979 A1) and 28F11 (U.S. Pat. No. 7,728,114 B2) are fully humanantibodies with specificity for the EGFRvIII tumor antigen and the humanCD3 complex, respectively. 28F11 has been described as the fully humananalog for the murine OKT3 clone, and was selected in this approach forits ability to bind CD3 and induce T cell activation through thisreceptor similarly to OKT3.

We subcloned the VH and VL genes from a fully human anti-EGFRvIII mAbdesignated “139,” and a fully human anti-CD3 activating mAb designated“28F11.” Through well-known techniques, we have constructed humansingle-chain Fvs by linking the VH and VL fragments of each antibodywith a (Gly4Ser): peptide linker. We have previously performed thesesame manipulations for the commercially available human CD3-specific mAbOKT3 and the murine anti-EGFRvIII mAb MR1-1 to manufacture the existingMR1-1xOKT3 BiTE described above. Using this technique, we have generateda fully human BiTE—thus reducing the potential generation ofneutralizing antibodies and permitting repeated administration—theexisting MR1-1xOKT3 construct could be further investigated clinicallyas an alternative given previous success in clinical trials with BiTEsderived from murine antibodies.

Cytotoxicity of the 139x28F11 construct will be measured by standardchromium-release assay using unstimulated human peripheral bloodlymphocytes (PBLs) as effector cells and human GBM cell lines U87MG andU87MG.ΔEGFR as target cells.

The xenograft system we used to obtain our preliminary data has thedistinct advantage of evaluating drug candidates for efficacy in ananimal system using human tumor tissue, with the potential to directlytranslate the therapeutic molecule of interest into clinical studies.However, a major drawback of this model is the lack of an endogenousimmune system, which drastically impedes our ability to appropriatelyevaluate safety and toxicity of our molecule, as well as performnumerous mechanistic studies that may only be appropriately performed insyngeneic hosts. Importantly, other T cell activating antibodies havehistorically met with unforeseen toxicity when translated in earlyclinical trials, at least in part due to the lack of appropriateimmunocompetent rodent models possessing surface molecules of equivalentbinding affinities and function to those found in humans (25).

The use of human CD3 transgenic mice in the preclinical evaluation ofour CD3-engaging BiTE, EGFRvIIIxCD3, is unusual. These mice have beenpreviously described to have ˜3 copies of the human CD3 transgeneintegrated chromosomally at unknown locations (26). When heterozygous,tgε600± mice possess near-normal numbers of peripheral T cells thatexpress both human and murine CD3. Notably, the tgε600± mouse strain hasbeen successfully used to test the function of anti-human CD3immunotoxins in preclinical models of T cell depletion and graftsurvival (27).

We have successfully imported the tgε600± mouse strain as embryos fromDr. Cox Terhorst (Beth Israel Deaconess Medical Center). We propose toestablish a colony of tgε600±, and using our preexisting murineEGFRvIII-expressing cell lines as targets, we will first examine theability for EGFRvIIIxCD3 to redirect splenocytes from tgε600± miceagainst tumor cells in vitro by standard chromium release assay. Afterestablishing minimum tumorigenic doses in the tgε600± mouse model, wewill proceed with a validation of the results obtained from experimentswe previously performed in the immunocompromised xenograft system.

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

-   1. Stupp R, Mason W P, van den Bent M J, et al: Radiotherapy plus    Concomitant and Adjuvant Temozolomide for Glioblastoma. New England    Journal of Medicine 352:987-996, 2005-   2. Phan G Q, Yang J C, Sherry R M, et al: Cancer regression and    autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4    blockade in patients with metastatic melanoma. Proceedings of the    National Academy of Sciences of the United States of America    100:8372-7, 2003-   3. Suntharalingam G, Perry M R, Ward S, et al: Cytokine storm in a    phase I trial of the anti-CD28 monoclonal antibody TGN1412. New    England Journal of Medicine 355:1018-28, 2006-   4. Bargou R, Leo E, Zugmaier G, et al: Tumor regression in cancer    patients by very low doses of a T cell-engaging antibody. Science    321:974-7, 2008-   5. Wikstrand C J, Hale L P, Batra S K, et al: Monoclonal antibodies    against EGFRvIII are tumor specific and react with breast and lung    carcinomas and malignant gliomas. Cancer Research 55:3140-8, 1995-   6. Baeuerle P A, Reinhardt C: Bispecific T-cell engaging antibodies    for cancer therapy. Cancer Research 69:4941-4, 2009-   7. Bigner S H, Humphrey P A, Wong A J, et al: Characterization of    the epidermal growth factor receptor in human glioma cell lines and    xenografts. Cancer Research 50:8017-8022, 1990-   8. Batra S K, Castelino-Prabhu S. Wikstrand C J, et al: Epidermal    growth factor ligand-independent, unregulated, cell-transforming    potential of a naturally occurring human mutant EGFRvIII gene. Cell    Growth & Differentiation 6:1251-1259, 1995-   9. Boockvar J A, Kapitonov D, Kapoor G, et al: Constitutive EGFR    signaling confers a motile phenotype to neural stem cells. Molecular    & Cellular Neurosciences 24:1116-30, 2003-   10. Lammering G, Hewit T H, Holmes M, et al: Inhibition of the type    III epidermal growth factor receptor variant mutant receptor by    dominant-negative EGFR-CD533 enhances malignant glioma cell    radiosensitivity. Clinical Cancer Research 10:6732-43, 2004-   11. Montgomery R B, Guzman J, O'Rourke D M, et al: Expression of    oncogenic epidermal growth factor receptor family kinases induces    paclitaxel resistance and alters beta-tubulin isotype expression.    Journal of Biological Chemistry 275:17358-63, 2000-   12. Archer G E, Sampson J H, Lorimer I A, et al: Regional treatment    of epidermal growth factor receptor vIII-expressing neoplastic    meningitis with a single-chain immunotoxin, MR-1. Clinical Cancer    Research 5:2646-52, 1999-   13. Yu X, Tsibane T, McGraw P A, et al: Neutralizing antibodies    derived from the B cells of 1918 influenza pandemic survivors.    Nature 455:532-6, 2008-   14. Smith, K., arman. L., Wrammert, J., Zheng. N.Y., Capra, J. D.,    Ahmed, R., and Wilson, P. C. 2009. Rapid generation of fully human    monoclonal antibodies specific to a vaccinating antigen. Nature    Protocols 4(3): 372-384.-   15. Heimberger A, Sun W, Hussain S, et al: Immunological responses    in a patient with glioblastoma multiforme treated with sequential    courses of temozolomide and immunotherapy. Neuro-Oncology 10:98-103,    2008-   16. Wrammert J, Smith K. Miller J. et al: Rapid cloning of    high-affinity human monoclonal antibodies against influenza virus.    Nature 453:667-71, 2008-   17. Wu X, Yang Z Y, Li Y, et al: Rational design of envelope    identifies broadly neutralizing human monoclonal antibodies to    HIV-1. Science 329:856-61, 2010-   18. Beers, R., Chowdhury, P., Bigner, D. and Pastan, I.    (Immunotoxins with increased activity against epidermal growth    factor receptor vIII-expressing cells produced by antibody phage    display. Clinical Cancer Research 6:2835-2843, 2000.-   19. Reddy S T, Ge X, Miklos A E, et al: Monoclonal antibodies    isolated without screening by analyzing the variable-gene repertoire    of plasma cells. Nature Biotechnology 28:965-9, 2010-   20. Kuan C T, Wikstrand C J, Archer G, et al: Increased binding    affinity enhances targeting of glioma xenografts by    EGFRvIII-specific scFv. International Journal of Cancer 88:962-9,    2000-   21. Sblattero D, Bradbury A. Exploiting recombination in single    bacteria to make large phage antibody libraries. NatureBiotechnology    18(1):75-80, 2000.-   22. Li B, Wang H, Dai J, et al: Construction and characterization of    a humanized anti-human CD3 monoclonal antibody 12F6 with effective    immunoregulation functions. Immunology 116:487-98, 2005-   23. Kuan C T, Reist C J, Foulon C F, et al: 125I-labeled    anti-epidermal growth factor receptor-vIII single-chain Fv exhibits    specific and high-level targeting of glioma xenografts. Clinical    Cancer Research 5:1539-49, 1999-   24. Heimberger A B, Learn C A, Archer G E, et al: Brain tumors in    mice are susceptible to blockade of epidermal growth factor receptor    (EGFR) with the oral, specific, EGFR-tyrosine kinase inhibitor    ZD1839 (iressa). Clinical Cancer Research 8:3496-502, 2002-   25. Attarwala H (2010) TGN1412: From Discovery to Disaster.    (Translated from eng) Journal of young pharmacists: JYP 2(3):332-336    (in eng).-   26. Wang, B. She, J., Salio, M., Allen, D., Lonberg, N., Terhorst,    C., Mol Med. 3:72-81, 1997.-   27. Weetall M, Digan M E, Hugo R, Mathew S, Hopf C, Tart-Risher N,    Zhang J, Shi V, Fu F, Hammond-McKibben D, West S, Brack R, Brinkmann    V, Bergman R, Neville D Jr, Lake P., T-cell depletion and graft    survival induced by anti-human CD3 immunotoxins in human CD3epsilon    transgenic mice. Transplantation. 2002 73:1658-66.

We claim:
 1. A bispecific polypeptide, comprising: a first single chainhuman variable region which binds to EGFRvIII, and comprises segmentsencoded by SEQ ID NO: 2 and 3; in series with a second single chainhuman variable region which binds to T cell activation ligand CD3 andcomprises segments encoded by SEQ ID NO: 5 and 6; wherein the bispecificpolypeptide comprises in N-terminal to C-terminal order segments encodedby SEQ ID NO: 2, 3, 4, 5, and
 6. 2. The bispecific polypeptide of claim1 which comprises segments encoded by SEQ ID NO: 2, 3, 4, 5, 6, and 7.3. A polynucleotide encoding the bispecific polypeptide of claim
 1. 4.The polynucleotide of claim 3 comprising the sequence of SEQ ID NO: 8.5. A method of treating a patient with an EGFRvIII-expressing tumor,comprising: administering the bispecific polypeptide of claim 1 to thepatient, whereby a cytolytic T cell response to the tumor is induced. 6.The bispecific polypeptide of claim 1 wherein each single chain variableregion comprises a disulfide bond between the V_(H) and the V_(L)domain.
 7. A method of making a bispecific polypeptide comprising:culturing a cell comprising the polynucleotide of claim 3 in a culturemedium such that it expresses the bispecific polypeptide, and collectingthe bispecific polypeptide from the cells or culture medium.